All hematopoietic lineages are derived from a pool of hematopoietic stem cells (HSCs) residing in the bone marrow. HSCs are characterized by their ability to self-renew to sustain the population, and differentiate to regenerate the hematopoietic system. The choice between self-renewal and lineage commitment is regulated by extrinsic and intrinsic factors, including epigenetic determinants such as DNA methylation. To directly address the role of de novo DNA methylation in HSCs, we generated a conditional knockout mouse model to show that loss of the DNA methyltransferase enzyme Dnmt3a impairs HSC differentiation and imposes a self-renewal program. However, the underlying molecular mechanisms remain obscure, and our group and others have not observed a correlation between altered DNA methylation patterns and gene expression changes. In this proposal, we are investigating epigenetic modifications outside of DNA methylation as the major effectors driving the phenotype. We have identified a critical set of regulatory genes that are safeguarded by the repressive chromatin mark H3K27me3 in normal HSCs, but lose this mark in Dnmt3a-null HSCs. We hypothesize that reduced repressive chromatin induces upregulation of HSC self-renewal genes in Dnmt3a-null HSCs, thereby impeding differentiation. Furthermore, our preliminary studies show that inhibition of the H3K27me3 demethylase Kdm6b can rescue many of the functional defects of Dnmt3a-null HSCs. The goal of this proposal is to understand how Dnmt3a and Kdm6b regulate gene expression in HSCs to maintain homeostatic balance between self-renewal and lineage commitment. We will test our hypothesis with the following specific aims using a combination of genetic mouse tools and clinically translatable agents; Specific Aim 1: Define the function of Kdm6b in HSC self-renewal. Specific Aim 2: Characterize the regulation of bivalent genes by Dnmt3a and Kdm6b. Specific Aim 3: The role of Kdm6b in Dnmt3a loss-of-function dysplastic hematopoiesis. Epigenetic dysregulation underlies many hematopoietic disorders, including myelodysplastic syndromes (MDS), a heterogeneous collection of hematopoietic diseases characterized by blood cell dysplasia and ineffective hematopoiesis. MDS arises from genetic mutations in HSCs that impair differentiation and lead to a clonal expansion in the bone marrow, and one of the most commonly mutated genes in MDS is DNMT3A. One of the long-term goals of the proposed studies is to identify targets for directed epigenetic therapies in DNMT3A-mutation patients (who have poor prognosis compared to DNMT3A wild-type patients) by understanding the underlying HSC dysfunction. As the outlook for MDS patients is poor (median survival varying from 5-months to 6-years depending on disease subtype), alternative treatment strategies are desperately needed. Given the increasing incidence of MDS in the population, these studies have a high significance for public health and the mission of the NIDDK.